A naloxonazine sensitive (mu1 receptor) mechanism in the parabrachial nucleus modulates eating

Abstract

The parabrachial nucleus (PBN) is an area of the brain stem that controls eating and contains endogenous opioids and their receptors. Previously, we demonstrated that acute activation of mu opioid receptors (MOPR) in the lateral PBN increased food consumption. MOPRs have been divided operationally into mu(1) and mu(2) receptor subtypes on the basis of the ability of naloxonazine (Nlxz) to block the former but not the latter. We used autoradiography to measure whether Nlxz blocks stimulation by the mu(1)/mu(2) agonist DAMGO (D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin) of the incorporation of [(35)S]-guanosine 5'(gamma-thio)triphosphate ([(35)S]-GTPgammaS) into sections of the PBN. In vitro, Nlxz dose dependently inhibited receptor coupling in all areas of the PBN. The 1 muM concentration of Nlxz reduced stimulation by 93.1+/-5% in the lateral inferior PBN (LPBNi) and by 90.5+/-4% in the medial parabrachial subregion (MPBN). Administration of Nlxz directly into the LPBNi decreased both food intake and agonist stimulated coupling, ex vivo, for the 24-h period after infusion. Infusion of Nlxz into the intended area reduced food intake by 42.3% below baseline values. Nlxz infusion prevented DAMGO stimulation of G-protein coupling in LPBNi and markedly reduced this stimulation in the MPBN. The incomplete inhibition of DAMGO-stimulated coupling in the MPBN is most likely due to the limited diffusion of Nlxz from the site of infusion (LPBNi) into this brain region. In conclusion, this study demonstrates that the mu(1) opioid receptor subtype is present in the parabrachial nucleus of the pons and that these receptors serve to modulate feeding in rats.

Fig. 1. The selective µ₁ antagonist Nlxz decreased in vitro stimulation of G-protein coupling by DAMGO (1µM) in the PBN

Data represent stimulation by DAMGO above basal values (fmol/g means±SEM). Asterisks indicate difference from value for DAMGO without Nlxz pretreatment: ** p<0.01 for MPBN and LPBNi, *p<0.05 for LPBNi (F(5,5)=13.7). Student Newman-Keuls test after ANOVA (n=7).

Fig. 2. Schematics showing the location of PBN in dorsal pons (left panel) and subregions sampled for autoradiography (right upper panel)

The line drawing shown in left panel is at the coronal level, approximately 9.8mm caudal to bregma according to the Paxinos and Watson atlas (1998). The upper right panel shows the inferior aspect of the lateral inferior PBN (LPBNi), which includes two regions of interest within the central subregion and one within the external subregion of this nucleus (C1, C2 and LE, respectively). The medial PBN (MPBN) includes a central and external subregion (MC and ME, respectively). Boxes represent the anatomical subregions of the PBN, which were used for densitometric analysis of G-protein coupling, as described in the methods section. Infusions (black filled circle) were made in the LPBNi. Open triangles represent infusions that were outside of the targeted area.

Fig. 3. Infusion of Nlxz reduced consumption of food

Data separated on the basis of infusions within the intended region of the LPBNi (Saline-in and Nlxz-in, both with n=7) or outside the intended region (Saline-out and Nlxz out, both with n=3). Values are presented as mean±SEM. Asterisks (**) represent difference from their respective baseline (BL) values, F(3,22)= 10.4, p<0.01.

Fig. 4. GTPγS DAMGO incorporation after saline or Nlxz infusion

DAMGO increased GTPγS incorporation ex vivo in saline infused rats (DAMGO-saline). Infusion of Nlxz prevented this response (DAMGO-Nlxz). Bottom panels show G-protein coupling when no drug (basal) or unlabeled GTPγS was present (non-specific, NS).

Fig. 5. Naloxonazine-infused animals prevented or reduced GTPγS DAMGO incorporation on the LPBNi and MPBN, respectively

At the end of the 24h measurements of food intake, five rats from Saline-in group, six from Nlxz-in and three from each of the Saline-out and Nlxz-out groups were sacrificed to quantify the effects of Nlxz on the ability of DAMGO to stimulate [35S]GTPγS incorporation into LPBNi and MPBN subregions. Infusion of Nlxz significantly decreased DAMGO stimulated G-protein coupling in LPBNi. A smaller but significant reduction occurred in MPBN. Data expressed as [35S]-GTPγS incorporation in fmol/g stimulation above basal. Asterisks (**) represent difference in DAMGO stimulation between Nlxz or saline groups. F(2,27)=3.2 (MPBN) and F(2,27)=10.6 (LPBNi), both p<0.001.